This proposal will determine how the intestinal microbiota shapes the development of innate-like B cells and the specificities of antibodies represented in the natural IgM (nIgM) repertoire. nIgM plays significant roles in maintenance of immunological homeostasis and the clearance of potentially immunogenic autoantigens represented on apoptotic cells, suppressing the development of autoimmune and allergic diseases. Innate-like B cells are the source of nIgM, and possess specificities for polysaccharide and phospholipid epitopes that are conserved between mammalian antigens and commensal bacteria harbored within the gut, however little is known regarding the impact of microbiota-derived antigens on the development of this subset of antibodies. Observations that innate-like B cells undergo antigen selection in the small intestine lamina propria leading to the differentiation of IgA secreting plasma cells in those tissues, and observations that mice maintained under germ free conditions experience exacerbated diseases in allergy models and in several autoimmune disease models led us to hypothesize that the microbiota plays a previously unappreciated role in shaping the systemic B cell repertoire and the generation of nIgM. We investigated the distribution of B cells specific for the T cell-independent (TI) antigen N-acetyl-D-Glucosamine (GlcNAc) between specific pathogen free and germ free mice and have found substantial evidence that intestinal colonization increases the numbers of B cells and levels of serum antibody specific for GlcNAc, while inducing shifts in the dominant immunoglobulin V-gene used to generate this specificity. To further dissect these interactions, we developed a novel fluorescence-activated cell sorting approach to study the distribution of TI antigens on species of commensal bacteria. Through simultaneous evaluation of the IgA opsonization status of these antigens, we expect to (i) identify immunologically relevant microorganisms bearing specific TI- antigens that interface with humoral immunity in the gut. Using gnotobiotic animal models, wherein germ free mice are reconstituted with flora enriched for particular antigenic determinants with the platform described above, we plan to test the hypothesis that antigen derived from the commensal micoflora leads to selection of B cell clonotypes into the nIgM-producing niche. We plan to test this hypothesis by (ii) evaluating the frequency and phenotype of splenic GlcNAc and Lewis X antigen-specific B cells following microbial reconstitution with bacteria commensal isolates enriched for these antigens, (iii) monitoring the generation of LP, spleen and bone marrow-localized plasma cells, and (iv) monitoring shifts in the B cell clonotypes composing those specificities. These experiments will demonstrate for the first time that interaction with exogenous antigen at peripheral tissues is required for development of a fully competent nIgM repertoire. This work will lead to the development of novel means of classifying the diversity of the intestinal microbiota, and offer valuable insight to the development of the nIgM repertoire value in designing immunotherapies aimed at boosting natural immunity.